mAbs最新文献

{"title":"Development of single-chain C1q affinity chromatography-mass spectrometry for the glycoform-resolved characterization of low-affinity immunoglobulin G interactions.","authors":"Steffen Lippold, Kelvin Chang, Mirjam Dürkoop, Jan Terje Andersen, Feng Yang, Tilman Schlothauer","doi":"10.1080/19420862.2026.2687922","DOIUrl":"10.1080/19420862.2026.2687922","url":null,"abstract":"<p><p>The interaction between complement factor C1q and the Fc region of immunoglobulin G (IgG) antibodies is modulated by the conserved IgG glycosylation site at Asn 297 and is a critical determinant of complement-dependent cytotoxicity. However, characterizing this weak, monovalent interaction is challenging, as standard binding assays fail to resolve the inherent heterogeneity of antibody proteoforms without specific engineering or enrichment. Here, we report the development of a single-chain C1q affinity chromatography-mass spectrometry (scC1q AC-MS) method to resolve IgG glycoforms. A major hurdle in analyzing low-affinity interactions is nonspecific binding (NSB) to the stationary phase. Through systematic screening of volatile salts, we identified ammonium hydrogencarbonate as a suitable mobile phase, significantly reducing NSB compared to standard ammonium acetate or formate solvents and previously established nonvolatile buffers. The optimized method successfully reproduced the established affinity hierarchy of human IgG subclasses (IgG3 > IgG1 ≫ IgG2 >IgG4). Furthermore, high-resolution MS detection enabled the separation of individual glycoforms, confirming that high-mannose glycans significantly reduce C1q affinity, whereas galactosylation provides a modest enhancement. The novelty of our developed method lies in advancing scC1q AC into an MS-compatible platform, which eliminates the need for laborious glycoengineering by providing a direct, simultaneous binding readout of individual glycoforms within heterogeneous antibody proteoform mixtures. By enabling the unbiased comparison of intrinsic antibody affinities independent of glycosylation variability, scC1q AC-MS provides a powerful new tool for the development and characterization of IgG antibodies and establishes a blueprint for the chromatographic analysis of weak protein interactions.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2687922"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13285597/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148277819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of afucosylation strategy on antibody function: a comparative study of glycoengineered anti-CD20 antibodies Obinutuzumab and Obinutuzumab beta.","authors":"Qing Shuang, Mingze Sun, Guangzhong Lin, Lunfeng Zhang, Shuo Huang, Qunfang Shen, Lingling Jiang, Lijing Yang, Jicui Sun, Hong Chen, Feng Li, Jiangmei Li","doi":"10.1080/19420862.2026.2657099","DOIUrl":"10.1080/19420862.2026.2657099","url":null,"abstract":"<p><p>Enhancing antibody-dependent cellular cytotoxicity (ADCC) via N-glycan afucosylation of Asn-297 is a validated strategy to improve the clinical efficacy of therapeutic antibodies. However, the impact of distinct glycoengineering approaches on the function of antibodies has not been systematically elucidated. Here, we experimentally compared two type II anti-CD20 antibodies, Obinutuzumab (Gazyva®) and Obinutuzumab beta (MIL62, Bejescin®), which share identical amino acid sequences but exhibit divergent glycosylation profiles. MIL62 was engineered with complete core afucosylation (fucose < 0.1%) lacking bisecting N-acetylglucosamine (GlcNAc) via knockout of the GDP-fucose transporter (GFT) in Chinese hamster ovary (CHO) cells used to produce the antibody. Conversely, Gazyva was produced in CHO cells that overexpress β-1,4-N-acetylglucosaminyltransferase III (GnT-III) and α-mannosidase II (α-ManII), resulting in ~50% fucose content and >80% bisecting GlcNAc occupancy. These distinct glycoengineering strategies led to disparate functional outcomes: afucosylated MIL62 showed improved FcγRIIIA binding and ADCC potency, while Gazyva with bisecting GlcNAc modifications exhibited higher glycoform heterogeneity and reduced thermal stability. Both antibodies displayed comparable FcRn binding, mannosylation, sialylation, and murine pharmacodynamics, mediating complete depletion of B cells in blood, lymph nodes, and spleen. Upon antigen rechallenge, MIL62 suppressed specific antibody titers, indicating memory B-cell eradication and profound potential to prevent autoimmune relapse. This study demonstrates that distinct glycoengineering strategies fundamentally reshape the antibody glycan profile beyond merely reducing fucose. These structural differences not only fine-tune ADCC potency, but also impact antibody stability. Particularly, complete afucosylation of MIL62 led to optimized ADCC potency and effectively suppressed pathogenic B cells repopulation in a delta-like ligand 3 (DLL3) re-challenge model, providing a critical framework for designing next-generation antibodies with superior therapeutic efficacy.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2657099"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13078206/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147674411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient brain delivery and sustained retention of antibodies via dual targeting of blood-brain barrier and intra-brain proteins.","authors":"Yuki Noguchi, Naoka Hironiwa, Kazuki Sato, Masaki Yamazaki, Masashi Kawashima, Nishiki Nagaya, Keitaro Nakagawa, Momoko Okuda, Atsuhiko Kato, Feng Shu, Kenta Haraya, Taichi Kuramochi","doi":"10.1080/19420862.2026.2724587","DOIUrl":"10.1080/19420862.2026.2724587","url":null,"abstract":"<p><p>Efficient delivery of therapeutic antibodies to the brain has been increasingly achieved using receptor-mediated transcytosis (RMT) approaches such as transferrin receptor (TfR)-mediated shuttle systems. Moreover, a strategy for enhancing brain retention has recently emerged as a focus of attention to overcome the rapid clearance of TfR shuttles from the brain. Here, we propose a novel approach that combines binding to intra-brain proteins, such as myelin oligodendrocyte glycoprotein (MOG), cell adhesion molecule 3, and chondroitin sulfate proteoglycan 5, for sustained retention in the brain with binding to blood-brain barrier (BBB) proteins, such as TfR and insulin-like growth factor 1 receptor, for enhanced BBB permeability. A mouse pharmacokinetic study demonstrated that the anti-MOG/TfR antibody reached a higher maximum concentration and maintained higher concentrations in the brain for 4 months than anti-MOG and anti-TfR antibodies. In immunohistochemistry and brain 3D-imaging study, the anti-MOG/TfR antibody distributed throughout the whole brain, suggesting it penetrates the BBB across the whole brain parenchyma and is retained there. Finally, this antibody technology was applied to brain delivery of neprilysin, an enzyme that degrades amyloid beta, demonstrating that it can enhance both the brain exposure and the pharmacodynamic effect of the potential therapeutic molecule. In conclusion, the concept of achieving sustained high brain exposure by combining BBB transport with enhanced brain retention was shown to be broadly applicable. This novel antibody technology platform is expected to deliver and retain various therapeutic molecules efficiently within the brain, addressing critical challenges in drug development for the central nervous system.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2724587"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13532014/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148850958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tangential flow filtration as a novel approach for fluorescent tracer purification and production up-scaling: cetuximab-800CW and ustekinumab-800CW as a proof-of-concept.","authors":"Henrik K Huizinga, Rosalie J van Dijken, Bahez Gareb, Bart G J Dekkers, Jacoba van Zanten, Hans H Westra, Pauline Koopmans, Wouter B Nagengast, Marjolijn N Lub-de Hooge","doi":"10.1080/19420862.2026.2743444","DOIUrl":"10.1080/19420862.2026.2743444","url":null,"abstract":"<p><p>Fluorescence molecular imaging using fluorescently labeled antibodies enables identification of target cells and provides pharmacological insight into local drug distribution. The current GMP manufacturing method of tracers using PD-10 columns is subject to methodological constraints when handling low antibody concentrations, large quantities, or incompatible antibodies. This study presents Tangential Flow Filtration (TFF) as a novel and scalable approach to manufacture clinical-grade fluorescent tracers and demonstrates its applicability using cetuximab-800CW and ustekinumab-800CW. TFF system settings were optimized for purification of cetuximab-800CW and ustekinumab-800CW with labeling parameters and quality control similar to tracer production with PD-10 columns. Cetuximab-800CW production was successfully transitioned from PD-10 to TFF. QC-data after manufacturing with TFF (<i>n</i> = 7) or PD-10 (<i>n</i> = 21) were compared at release and up to 24 months of storage at 2-8°C. This revealed TFF purification to be more efficient and robust, with label efficiencies of 93.5 ± 2.3% with TFF compared to 68.3 ± 6.9% with PD-10, and 0.0 ± 0.1% unconjugated dye with TFF compared to 0.5 ± 0.3% with PD-10. Stability data were comparable for both methods. TFF manufacturing yielded clinical-grade cetuximab-800CW batches stable for at least 24 months. Concentrations ranged from 0.99-1.08 mg/mL and target binding affinity from 65-98%. Detected free dye and aggregates remained well below 3.0%. Moreover, ustekinumab-800CW was successfully manufactured using TFF after PD-10 manufacturing failed due to particle formation. Ustekinumab-800CW was stable up to 24 months at 2-8°C. Together, this demonstrates the feasibility of using TFF for clinical-grade fluorescent tracer manufacturing under GMP conditions.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2743444"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13652071/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149137420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hybrid AI/ML-mechanistic framework enables intelligent optimization of commercial biopharmaceutical downstream processing.","authors":"Rong-Zhu Wang, Yu-Xin Liao, Teng-Long Wang, Xiao-Yang Jing, Guang Yang, Hao Wang, Feng Xiao, Yi Zong, Yan Yang, Ye Yuan, Xuan Xian, Shan-Jing Yao, Lei Jin, Dong-Qiang Lin, Yun Kenneth Kang","doi":"10.1080/19420862.2026.2644662","DOIUrl":"10.1080/19420862.2026.2644662","url":null,"abstract":"<p><p>Biopharmaceutical manufacturing requires continuous improvement to ensure robust, efficient, and high-quality processes, yet traditional experimental designs remain resource-demanding and insufficient to capture interactions of multiple parameters. Here, we introduce a hybrid framework integrating artificial intelligence (AI)/machine learning (ML) with mechanistic modeling to optimize anion-exchange chromatography and resolve the long-standing yield-purity trade-off in PEGylated protein purification. Three critical process parameters were first identified through correlation analysis between 30 input factors and critical quality attributes/process yield from 400+ commercial manufacturing lots, which were further refined using equilibrium dispersive and steric mass action models. Over 40,000 <i>in silico</i> optimization via the mechanistic model resolved the yield-purity trade-off, achieving a 12% increase in yield and 33% reduction in high-molecular-weight impurities. The optimized process conditions were verified across laboratory (<i>n</i> = 3), pilot (<i>n</i> = 3), and commercial (<i>n</i> = 18) runs, consistently demonstrating scalability and process robustness. This study highlights the power of combining data-driven machine learning with mechanistic modeling for process optimization, leading to an improved commercial process with substantial cost savings and paving the way for upcoming intelligent biomanufacturing.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2644662"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13020864/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147513336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Self-interaction nanoparticle spectroscopy predicts high-concentration viscosity of therapeutic IgG1 antibodies.","authors":"Santosh Kumar Paidi, Julissa Ibrahim, Kateryna Stepurska, Jonathan Zarzar, Saeed Izadi, Erina Rude, Steven Luu, Daniel Kovner, Kelly O'Connor, Karenna Bol, Shrenik Mehta, Nisana Andersen, Nicole Stephens, Emily Makowski, Joel Heisler, Trevor Swartz, Paul J Carter, Tomasz Baginski","doi":"10.1080/19420862.2026.2709948","DOIUrl":"10.1080/19420862.2026.2709948","url":null,"abstract":"<p><p>Predicting high-concentration viscosity of monoclonal antibodies is crucial for their development as therapeutics for subcutaneous delivery, but traditional experimental rheometry methods for assessing viscosity are low-throughput, which limits their utility. This study evaluates self-interaction nanoparticle spectroscopy (SINS) assays - specifically charge-stabilized SINS (CS-SINS) and PEG-stabilized SINS (PS-SINS) - for high-throughput viscosity prediction. We characterized 96 IgG₁ antibodies, assessing SINS against <i>in silico</i> descriptors and dynamic light scattering (DLS) data. CS-SINS showed strong correlation with charge, offering limited additional utility. In contrast, PS-SINS provided orthogonal information; integrating it with <i>in silico</i> data and DLS significantly improved random forest model accuracy for binary viscosity classification. PS-SINS measurements in multiple buffers captured complementary information, achieving comparable accuracy without DLS. Importantly, PS-SINS scores exhibited a strong logarithmic relationship with high-concentration viscosity in Fc variants of clinical antibodies (<i>r</i> = 0.72 and <i>r</i> = 0.85 for trastuzumab and omalizumab variants, respectively), suggesting a direct mechanistic link. Furthermore, PS-SINS performed reliably with one column-purified (protein A) samples, supporting its early-stage application. These findings establish PS-SINS as a high-throughput tool to accelerate the developability assessment of antibody candidates.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2709948"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13432892/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148630939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel bispecific T-cell engagers overcoming acquired EGFR resistance.","authors":"Lennart Kühl, Ann-Kathrin Löffler, Oliver Seifert, Dennis Michler, Miriam Kuhlmann, Giulio Russo, Philipp Kuhn, Helena Nowack, André Frenzel, Thomas Schirrmann, Monilola A Olayioye, Roland E Kontermann","doi":"10.1080/19420862.2026.2674236","DOIUrl":"10.1080/19420862.2026.2674236","url":null,"abstract":"<p><p>Epidermal growth factor receptor (EGFR) is a validated therapeutic target in several human cancers harboring wild-type KRAS. However, intrinsic and acquired resistance to EGFR-targeted antibody therapies such as cetuximab remains a major limitation to achieving broad and durable treatment responses. Extensive clinical and translational studies have shown that resistance frequently arises from missense mutations within the extracellular domain (ECD) of EGFR. In this study, we developed novel antagonistic EGFR antibodies that bind epitopes overlapping but distinct from the cetuximab-binding site while retaining high affinity for all major EGFR ECD escape variants. Antibody binding effectively inhibited EGFR phosphorylation, downstream signaling, and tumor cell proliferation. The antibodies were further engineered into bispecific EGFR × CD3 T-cell engagers (TCEs) with either 1 + 1 or 2 + 1 stoichiometries. In contrast to cetuximab-based TCEs, the newly developed EGFR-directed TCEs efficiently induced T cell-mediated cytotoxicity against tumor cells expressing wild-type EGFR as well as the clinically relevant ECD escape variants S492R and G465R. Anti-tumor activity was additionally demonstrated in an EGFR-expressing CT-26 syngeneic tumor model in vivo. Collectively, these findings define a promising therapeutic strategy to overcome resistance to current EGFR-targeted therapies and provide a strong rationale for the development of next-generation T-cell - redirecting therapies in patients with EGFR-positive malignancies.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2674236"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13192131/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147973554","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of ASO conjugation and receptor binding affinity on intracellular transport of mono- and bispecific TfR- and CD98-Brainshuttle^TM variants.","authors":"Tatjana Sela, Tamara Wirth, Annika Sommer, Vincent Larraillet, Joachim Butzer, Sabine Lohmann, Pia Wittmann, Christian Klein, Hendrik Knoetgen, Roberto Villasenor, Annette Indlekofer, Kerstin Hofer, Felix Schumacher, Jens Niewöhner","doi":"10.1080/19420862.2026.2691351","DOIUrl":"10.1080/19420862.2026.2691351","url":null,"abstract":"<p><p>Antisense oligonucleotides (ASOs) represent a promising therapeutic modality for central nervous system (CNS) disorders, offering highly specific modulation of gene expression. However, their clinical utility is severely limited by their inability to cross the blood-brain barrier (BBB), necessitating effective shuttling strategies. While transferrin receptor (TfR1)-mediated shuttling has shown therapeutic promise, the fundamental mechanisms governing the delivery of antibody-ASO conjugates across the BBB remain poorly understood. This study directly addresses this critical knowledge gap by establishing a mechanistic understanding of how the ASO cargo impacts major cellular interactions during the Brainshuttle^TM-mediated transport across the BBB. Using a panel of advanced in vitro assays developed specifically for this purpose, including quantitative transcytosis, detailed imaging-based intracellular trafficking, and binding assays with brain endothelial cells (BECs), the shuttling process was systematically investigated. We demonstrate that ASO conjugation profoundly alters the cellular fate of the Brainshuttle^TM. Specifically, conjugation increased the binding to BECs of low-affinity TfR1 shuttles via avidity effects while paradoxically reducing the binding strength of high-affinity shuttles. Functional assays confirmed the biological activity of the delivered ASOs; however, transcytosis of high-to-moderate affinity binders across the BBB model was significantly delayed upon ASO conjugation. Building on these mechanistic insights, we engineered TfR1 Brainshuttles^TM with optimized affinity and explored the shuttling potential of an alternative BBB receptor, CD98hc. These efforts culminated in the development of a novel bispecific Brainshuttle^TM targeting both CD98hc and TfR1. This dual-targeting strategy exploits distinct and potentially non-competing trafficking pathways to overcome ASO-induced delays and significantly enhance in vitro transcytosis efficiency. The in vitro findings in this study underscore the necessity of mechanism-driven design to overcome ASO-induced limitations in delivery across the BBB. The bispecific CD98/TfR1 approach presented here provides a promising new strategy for maximizing delivery efficiency and enabling more effective therapeutic outcomes for CNS diseases.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2691351"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13290098/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148302152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure-function analysis of empasiprubart, a calcium- and pH-dependent clinical phase complement C2 blocking antibody.","authors":"Laura Bracke, Heidi Gytz Olesen, Erwin Pannecoucke, Tim Delahaye, Emma K Persson, Christophe Blanchetot, Hans de Haard, C Erik Hack, Gregers Rom Andersen, Inge Van de Walle","doi":"10.1080/19420862.2026.2666430","DOIUrl":"10.1080/19420862.2026.2666430","url":null,"abstract":"<p><p>Empasiprubart (ARGX-117) is a humanized recycling antibody that prevents binding of C2 to C4b, blocking downstream classical and lectin pathways of complement activation. Empasiprubart binds to the CCP2 domain of complement component C2 in a calcium- and pH-dependent manner, leveraging physiological differences between blood and endosomal environments to facilitate the release and subsequent degradation of bound C2. The molecule incorporates Fc region mutations (H433K and N434F) that enhance its affinity for the neonatal Fc receptor (FcRn) under acidic endosomal conditions, thereby prolonging its <i>in vivo</i> half-life and supporting its recycling capacity. However, despite the earlier description of the complex structure, the molecular mechanism underlying these dependencies has remained elusive. Here, we further explored the crystal structure of the empasiprubart fragment antigen-binding (Fab) complexed to a C2 fragment, and provide a molecular rationale for its unique properties, while recognizing that not all contributing factors have been fully elucidated. Our observations indicate that the pH-dependent target release is rooted in a subtle intramolecular complementarity-determining region (CDR) destabilization, rather than direct modulation of the binding interface, and highlight the interplay between framework residues and CDRs. Collectively, our results not only lead to a better understanding of the mode of action of empasiprubart but also demonstrate the pivotal role of framework residues in the orchestration of antibody CDR function for non-trivial target binding.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2666430"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13154994/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147816829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of process impurities on developability assessments of complex antibody formats: the striking role of leachable copper.","authors":"Vincent Wieser, Christian Spick, Matthias Forster, Urs Hanke, Katja Montan, Michael Grote, Benjamin Weiche","doi":"10.1080/19420862.2026.2670845","DOIUrl":"10.1080/19420862.2026.2670845","url":null,"abstract":"<p><p>The assessment of developability and physical stability of antibody therapeutics is a cornerstone of modern biopharmaceutical discovery, increasingly relying on high-throughput automation to screen candidates to identify the most suitable lead candidate. In this study, we report a severe, anomalous fragmentation event observed in therapeutic antibodies during routine automated buffer exchange into phosphate-buffered saline (PBS) at physiological pH. Through a systematic investigation, we demonstrate that this instability is not intrinsic to the proteins, but is driven by the specific physicochemical environment of the purification process. We identify that at pH 7.4, the dominant phosphate species may act as a ligand capable of mobilizing trace catalytic copper from disposable chromatography columns. Our data suggests the leached copper coordinates to the antibody hinge region, driving a highly localized, Fenton-like oxidative cleavage. Comparative analysis reveals that this vulnerability is influenced by molecular architecture. Complex formats, such as 2 + 1 CrossMabs, exhibited significantly higher susceptibility than standard IgGs, suggesting that steric crowding may enhance the accessibility of the metal-binding site. Finally, we present a robust mitigation strategy using an ethylenediaminetetraacetic acid-based column conditioning protocol that effectively eliminates the leachable catalyst. These findings highlight a critical, often-overlooked source of chemical instability in automated workflows and underscore the necessity of controlling process-related impurities, particularly when developing complex antibody modalities.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2670845"},"PeriodicalIF":7.9,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13173997/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147930822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}